Cellulosic biofuel production aims to use microbes to convert sugars derived from plant matter into compounds like alcohols, alkenes, or cyclic hydrocarbons [3-5]. However, many biofuels are inherently toxic to microorganisms, limiting production potential. Microbial systems have several native strategies for dealing with fuel toxicity [2]. Some mechanisms, such as reducing membrane permeability or metabolizing hydrocarbons, are not ideal in a production strain because they may hinder endogenous biofuel production. A promising alternative uses membrane transport proteins to export biofuel [6].
Microbial metabolic pathways can be used to produce a variety of compounds that can serve as biofuels. Short to mid-length chain (C4-C12) alcohols such as butanol, isopentanol, and geraniol are bio-gasoline candidates [1, 3]. Longer chain compounds (C9-C23) such as geranyl acetate and farnesyl hexanoate are bio-diesel alternatives. In addition, cyclic alkenes like limonene and pinene serve as precursors to bio-jet fuel [1, 7]. While microbial biosynthetic routes to most of these compounds exist, these compounds have known antimicrobial activity.
Efflux pumps are secondary transporters that can export toxins from the cell using the proton motive force [8-10]. In gram-negative bacteria they are composed of three protein homotrimers: an inner membrane protein—the proton antiporter that is responsible for substrate recognition, a periplasmic linker protein, and an outer membrane channel. All three proteins are essential for function and the corresponding genes are commonly arranged together in an operon.
A small number of efflux pumps have been previously characterized as solvent-resistant. Examples include ttgABC, ttgDEF, ttgGHI (toluene tolerance genes) [11, 12] and srpABC (solvent resistance pump) [13] from Pseudomonas putida. These pumps are induced by a range of compounds [14], but appear to be fairly specific to solvents [14, 15]. In addition, multidrug efflux pumps, like mexAB-oprM, mexCD-oprJ, and mexEF-oprN from Pseudomonas aeruginosa [16] and acrAB-tolC from E. coli [17], export a very broad range of substrates, including solvents. All efflux pumps in gram negative bacteria that have been characterized as solvent resistant fall into the hydrophobe/amphiphile efflux (HAE1) family of resistance-nodulation-division (RND) efflux pumps [18]; members of this family export a particularly wide range of toxic substrates, though not all pumps in this class export solvents [17]. Sequenced bacterial genomes encode a large number of HAE1 efflux pumps, and present a largely unexplored resource for discovering novel pumps with potential for use in engineering fuel tolerance.